The present invention relates to a new class of receptor protein tyrosine phosphatase molecule, the ligands that bind this new class of receptor, and the uses of such receptors and ligands. Specifically, the members of this new class of receptor protein tyrosine phosphatase molecule are proteoglycans and/or possess an extracellular carbonic anhydrase structural domain. The characterization of one member of this new class, RPTPxcex2, is described in the working examples presented herein. As is further demonstrated in the Working Examples presented below, the ligands which bind the receptor protein tyrosine phosphatases of the invention may be, for example, tenascin and/or members of the cell adhesion molecule (CAM) family of extracellular molecules.
Cells rely, to a great extent, on extracellular molecules as a means by which to receive stimuli from their immediate environment. These extracellular signals are essential for the correct regulation of such diverse cellular processes as differentiation, contractility, secretion, cell division, cell migration, contact inhibition, and metabolism. The extracellular molecules, which can include, for example, hormones, growth factors, or neurotransmitters, act as ligands that bind specific cell surface receptors. The binding of these ligands to their receptors triggers a cascade of reactions that brings about both the amplification of the original stimulus and the coordinate regulation of the separate cellular processes mentioned above.
A central feature of this process, referred to as signal transduction (for recent reviews, see Posada, J. and Cooper, J. A., 1992, Mol. Biol. Cell 3:583-592; Hardie, D. G., 1990, Symp. Soc. Exp. Biol. 44:241-255), is the reversible phosphorylation of certain proteins. The phosphorylation or dephosphorylation of amino acid residues triggers conformational changes in regulated proteins that alter their biological properties. Proteins are phosphorylated by-protein kinases and are dephosphorylated by protein phosphatases. Protein kinases and phosphatases are classified according to the amino acid residues they act on, with one class being serine-threonine kinases and phosphatases (reviewed in Scott, J. D. and Soderling, T. R., 1992, 2:289-295), which act on serine and threonine residues, and the other class being the tyrosine kinases and phosphatases (reviewed in Fischer, E. H. et al., 1991 Science 253:401-406; Schlessinger, J. and Ullrich, A., 1992, Neuron 9:383-391; Ullrich, A. and Schlessinger, J., 1990, Cell 61:203-212), which act on tyrosine residues. The protein kinases and phosphatases may be further defined as being receptors, i.e., the enzymes are an integral part of a transmembrane, ligand-binding molecule, or as non-receptors, meaning they respond to an extracellular molecule indirectly by being acted upon by a ligand-bound receptor. Phosphorylation is a dynamic process involving competing phosphorylation and dephosphorylation reactions, and the level of phosphorylation at any given instant reflects the relative activities, at that instant, of the protein kinases and phosphatases that catalyze these reactions.
While the majority of protein phosphorylation occurs at serine and threonine amino acid residues, phosphorylation at tyrosine residues also occurs, and has begun to attract a great deal of interest since the discovery that many oncogene products and growth factor receptors possess intrinsic protein tyrosine kinase activity. The importance of protein tyrosine phosphorylation in growth factor signal transduction, cell cycle progression and neoplastic transformation is now well established (Cantley, L. C. et al., 1991, Cell 64:281-302; Hunter T., 1991, Cell 64:249-270; Nurse, 1990, Nature 344:503-508; Schlessinger, J. and Ullrich, A., 1992, Neuron 9:383-391; Ullrich, A. and Schlessinger, J., 1990, Cell 61:203-212). Subversion of normal growth control pathways leading to oncogenesis has been shown to be caused by activation or overexpression of tyrosine kinases which constitute a large group of dominant oncogenic proteins (reviewed in Hunter, T., 1991, Cell 64:249-270).
In addition, since the initial purification, sequencing and cloning of a protein tyrosine phosphatase (Thomas, M. L. et al., 1985, Cell 41:83), additional potential protein tyrosine phosphatases have been identified at a rapid pace. (See, for example, Kaplan, R. et al., 1990, Proc. Natl. Acad. Sci. USA 87:7000-7004; Krueger, N. X. et al., 1990, EMBO J. 9:3241-3252; Sap, J. et al., 1990, Proc. Natl. Acad. Sci. USA 87:6112-6116). Because the number of different protein tyrosine phosphatases that have been identified is increasing steadily, speculation has arisen that the protein tyrosine phosphatase family may be as large as the protein tyrosine kinase family (Hunter, T., 1989, Cell 58:1013-1016). With this increase in the reported cloning of protein tyrosine phosphatase genes, the role that the regulation of dephosphorylation may have in the control of cellular processes has also begun to receive more attention.
As mentioned above, protein tyrosine phosphatases (PTPases) can be classified into two subgroups, the non-receptor and receptor classes. The non-receptor class is composed of low molecular weight, cytosolic, soluble proteins. All known non-receptor PTPases contain a single conserved catalytic phosphatase domain of approximately 230 amino acid residues. (See, for example, Charbonneau et al., 1989, Proc. Natl. Acad. Sci. USA 86:5252-5256; Cool et al., 1989, Proc. Natl. Acad. Sci. USA 86:5257-5261; Guan et al., 1990, Proc. Natl. Acad. Sci. USA 87:1501-1502; Lombroso et al., 1991, Proc. Natl. Acad. Sci. USA 88:7242-7246.) Sequence analysis reveals that about 40 of the amino acids of the catalytic domain are highly conserved, and a very highly conserved segment of 11 amino acid residues with the consensus sequence [I/V]HCXAGXXR[S/T]G, is now recognized to be a hallmark of the protein tyrosine phosphatase catalytic domain.
The receptor class is made up of high molecular weight, receptor-linked PTPases, termed RPTPases. Structurally resembling growth factor receptors, RPTPases consist of an extracellular, putative ligand-binding domain, a single transmembrane segment, and an intracellular catalytic domain (reviewed in Fischer et al., 1991, Science 253:401-406). The intracellular segments of almost all RPTPases are very similar. These intracellular segments consist of two catalytic phosphatase domains of the type described above, separated by an approximately 58 amino acid residue segment. This two domain motif is usually located approximately 78 to 95 amino acid residues from the transmembrane segment and is followed by a relatively short carboxy-terminal amino acid sequence. The only known exception is the isoform HPTPxcex2 (Krueger, N. X. et al., 1990, EMBO J. 9:3241), which contains only one catalytic phosphatase domain.
While the intracellular RPTPase segments are remarkably highly conserved, the RPTPase extracellular domains are highly divergent. For example, certain RPTPases possess a heavily glycosylated external domain and a conserved cysteine-rich region (Thomas, M. L. et al., 1985, Cell 41:83; Thomas, M. L. et al., 1987, Proc. Natl. Acad. Sci. USA 84:5360; Ralph, S. J. et al., 1987, EMBO J. 6:1251-1257) while others contain immunoglobulin G-like (Ig) domains linked to fibronectin type III repeats (Streuli, M. et al., 1989, Proc. Natl. Acad. Sci. USA 86:8698; Streuli, M. et al., 1988, J. Exp. Med. 168:1523). Still other RPTPases contains only multiple fibronectin type III repeats (Krueger, N. X. et al., 1990, EMBO J. 9:3241), while certain RPTPases have smaller external domains that contain several potential glycosylation sites (Jirik, F. R. et al., 1990, FEBS Lett. 273:239). The ligands that regulate RPTPs have not been identified. It has been speculated that circulating extracellular factors are unlikely to bind to those receptors containing Ig and/or fibronectin Type III repeats and that interaction with other surface antigens, perhaps on other cells, is more likely to be the case with these receptors.
Because enhanced tyrosine phosphorylation has been shown to be responsible for causing cellular transformation, underexpression, or inactivation, of protein tyrosine phosphatases may also potentially result in oncogenesis. For this reason, tyrosine-specific phosphatase genes are candidate recessive oncogenes or tumor suppressor genes. In support of this theory, the human RPTPase, RPTPxcex3, has been shown to map to a chromosomal region, 3p14-21, which is frequently deleted in renal cell and lung carcinomas (LaForgia, S. et al., 1991, Proc. Natl. Acad. Sci. USA 88:5036-5040). Recent studies, however, indicate that protein tyrosine phosphatase action need not only be suppressive. It has been shown that members of the src family of non-receptor tyrosine kinases contain inhibitory tyrosine phosphorylation sites in the carboxy terminal tails (reviewed by Hunter, T., 1987, Cell 49:1-14). When these sites are phosphorylated, the molecules"" tyrosine kinase activity is inhibited (Nada, S. et al., 1991, Nature 351:69-72). It has further been demonstrated that, in T cells, the dephosphorylation of such inhibitory sites by a protein tyrosine phosphatase (CD45) leads to enhanced tyrosine phosphorylation (Ledbetter, J. A. et al., 1989, Proc. Natl. Acad. Sci. USA 86:8628-8632), indicating, therefore, that phosphatases may function as activating and well as inhibitory signaling enzymes. Also, dephosphorylation of a tyrosine residue has been suggested to be an obligatory step in the mitotic activation of the maturation-promoting factor kinase (Morla, A. O. et al., 1989, Cell 58:193-203). Taken together, the above observations suggest that PTPases may play an important role in cellular control mechanisms, as effectors in mechanisms of transmembrane signaling, as cell-cycle regulators, and as potential oncogenes and anti-oncogenes.
The present invention relates to a new class of receptor protein tyrosine phosphatase molecule, to the family of ligands that binds this new class of receptor, and to the uses of such receptors and ligands. Specifically, the members of this new class of receptor protein tyrosine phosphatase molecule are proteoglycans and/or possess an extracellular carbonic anhydrase structural domain. The characterization of one such receptor molecule, RPTPxcex2, is described in the working examples presented herein.
The ligands which bind the receptor protein tyrosine phosphatases of the invention may include, but are not limited to, tenascin and/or members of the cell adhesion molecule (CAM) family of extracellular molecules. The discovery that CAMs bind receptor protein tyrosine phosphatases represents the first identification of a natural ligand for this type of receptor. Binding of two CAMs, namely N-CAM and Ng-CAM, to the receptor protein tyrosine phosphatases of the invention is demonstrated in the working examples presented herein. In addition, as is demonstrated in the Working Example presented below in Section 8, the receptor protein tyrosine phosphatases of the invention also bind the extracellular matrix molecule tenascin. The receptors and the receptor-binding ligands of the invention may be used to develop compounds and strategies for modulating cellular processes under the control of the receptor protein tyrosine phosphatases. Such processes include, but are not limited to, normal cellular functions such as differentiation, metabolism, cell cycle control, wound healing and neuronal function; cellular behavior such as motility, migration, and contact inhibition, in addition to abnormal or potentially deleterious processes such as virus-receptor interactions, inflammation, cellular transformation to a cancerous state, and the development of Type 2, insulin Independent, diabetes mellitus. Compounds that may interfere with ligand binding are described and methods for identifying other potential ligands, such as CAM-type ligands, growth factors, or extracellular matrix components, are discussed.